Effect of Xanthohumol, a Bioactive Natural Compound from Hops, on Adenosine Pathway in Rat C6 Glioma and Human SH-SY5Y Neuroblastoma Cell Lines

Abstract

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer's disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A₁ and A₃, which inhibit the adenylyl cyclases (AC) pathway, and A_2A and A_2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A₁ and A_2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5'-nucleotidase, were analyzed. The adenosine A₁ receptor was significantly increased after Xn exposure, while no changes in A_2A receptor membrane levels or AC activity were reported. Regarding 5'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Keywords: adenosine receptors; cell cultures; xanthohumol.

Figure 1

Effect of Xn on viability and cell number of C6 (panel (A) top row and panel (B) and (D), respectively) and SH-SY5Y (panel (A) bottom row and panel (C) and (E), respectively) cell lines. Cells were treated with different concentrations of Xn, and cell viability was assayed. Histograms showing data, expressed as percentages with respect to control values (B,C) or the number of cells ×10⁵ (D,E), correspond to the mean ± SEM of three different experiments carried out in sextuplicate. Individual data are shown in circles, squares and triangles. * p < 0.05 and ** p < 0.01 are significantly different from the control values according to the Student’s t-test.

Figure 2

Gene expression of A₁ (panel (B)) and A_2A (panels (C,D)) adenosine receptors and AC1 enzyme isoform (panel (A)) in C6 and SH-SY5Y cell lines. The expression of the β-actin gene was used as an endogenous control for each sample. Histograms show data corresponding to the mean ± SEM of three–four different experiments performed in duplicate. Individual data are shown in circles and squares. * p < 0.05 is significantly different according to the Student’s t-test.

Figure 3

Adenosine A₁ and A_2A receptor levels in plasma membranes from C6 and SH-SY5Y after Xn exposure. Western blot analysis was performed with plasma membranes obtained after Xn treatment for 24 h for A₁R (panel (A) and (B) for C6 and SH-SY5Y cell lines, respectively) and A_2AR (panel (C,D) for C6 and SH-SY5Y cell lines, respectively). Graphs representing data, expressed as % from control values, correspond to the mean ± SEM of four experiments carried out with different plasmatic membrane isolations normalized using β-actin as the control loading. Individual data are shown in circles and squares.** p < 0.01 is significantly different from the control according to the Student’s t-test.

Figure 4

Adenylate cyclase activity in plasmatic membrane fractions from C6 and SH-SY5Y cells. Adenylate activity was analyzed in plasma membranes after Xn treatment for 24 h. Histograms show data representing basal (panels (A,D)), forskolin-stimulated (panels (B–E)), and CPA inhibited over forskolin-stimulated activity (panels (C,F)) in C6 and SH-SY5Y cell lines, respectively. Data are expressed as pmol cAMP/mg protein·min for basal activity, as a percentage of basal values for forskolin-stimulated, and as % from forskolin-stimulated for CPA-inhibited activity, and they correspond to the mean ± SEM of three–four experiments performed using different plasma membrane isolations. Individual data are shown in circles and squares. Dashed line in B, E correspond to basal value, set to 100 %; dashed line in C, F correspond to forskolin-stimulated value, set to 100 % * p < 0.05 and ** p < 0.01 are significantly different from basal (panels (B,E)) or forskolin-stimulated activity (panels (C,F)) according to the Student’s t-test.

Figure 5

PKA levels in C6 (panel (A)) and SH-SY5Y (panel (B)) cells. Western blots were performed in cytosol fractions from cells after 10 µM Xn treatment for 24 h. Histograms show that results, expressed as % of control values, correspond to the mean ± SEM of four experiments performed using different cytosol fractions and normalized using GAPDH as the control loading. Individual data are shown in circles and squares. No differences were found according to the statistical analysis.

Figure 6

5′-Nucleotidase enzymatic activity in the plasmatic membrane (CD73) and cytosol fractions from C6 (panels (A,C)) and SH-SY5Y (panels (B,D)) cell lines, respectively, after 10 µM Xn exposure for 24 h. Results are expressed as the mean ± SEM of four different experiments performed in duplicate. Individual data are showin in circles and squares. * p < 0.05 is significantly different according to the Student’s t-test.